Introduction. Burkholderia pseudomallei (BP) is a prime candidate for use in biowarfare and bioterrorism because it can be easily obtained (from the environment in endemic areas), propagated and prepared (robust organism rapidly growing on simple media), easily dispersed with high infectivity (aerosolization and contamination of water), and causes a rapidly developing, severe disease (melioidosis) with high mortality, even when properly diagnosed. Treatment of melioidosis is greatly complicated due to BP's intrinsic resistance to most classes of antibiotics. Current therapies are expensive and include 10 day intravenous administration of cephalosporins, followed by oral eradication therapy with a regimen of two to four drugs for 3-5 months. Infections of a large number of people would therefore be disastrous in terms of financial and human costs. In this project we will characterize efflux pumps of the resistance nodulation and cell division (RND) family, the suspected main players in BPs intrinsic and acquired antibiotic resistance. Using powerful molecular and genetic tools, we will characterize and assess the clinical significance of these pumps and construct strains that each express a single efflux pump as a tool for efflux pump inhibitor (EPI) discovery. We will then screen compound libraries for EPIs, with the project goal being the discovery of EPI lead compounds. It is hoped that these efforts will ultimately provide new therapeutic approaches for melioidosis by increasing the effectiveness of existing drug therapies, and by enabling the use of cheaper and readily available drugs. Project interactions. This project II.A.4 (Schweizer, PI) will have significant interactions with other RCE projects and core facilities. Genetic tools developed in this project will lead to obvious interactions with other projects involving Burkholderia ssp. and its mutants, including II.C.1, Fur-regulated genes (Vasil, PI), II.C.2, oxidative and nitrosative stress (Voskuil, PI), II.C.3, O2-dependent host defenses (Vazquez-Torres, PI), II.C.4, iron uptake (Holmes, PI) and II.A.1, role of innate immunity in pulmonary infection (Dow and Bosio, Co-Pis). Efflux mutants and pump overexpressing strains will be powerful tools for the drug discovery efforts initiated in project M.A.2., anti-infectives against select agents (Crick and Brennan, Co-Pis). The Select Agents Archive Core III.D (Robison, Director) will provide bacterial strains and clinical isolates; and the Genomics/Proteomics Core III.C. (Slayden, Director) will provide HTS screening facilities, and access to compound libraries and microarrays.